Arrestin is a cytosolic protein that serves to control G-protein Coupled Receptors (GPCRs) by mediating downregulation of GPCR function upon prolonged exposure to a ligand. G-protein coupled receptors are the targets for many of the most notorious prescription pharmaceuticals that lead to addiction such as several of the opiod analgesics (e.g. Vicodin, Oxycontin) in addition to illicit drugs (e.g. cannabis). This downregulation of the GPCR is a factor leading to drug tolerance which is a key issue in understanding addiction. Following activation by a ligand, GPCRs are phosphorylated at the C-terminus which allows the formation of a complex with arrestin. Arrestin is a scaffold protein that can then recruit other protein binding partners and this leads to desensitization and internalization of the GPCR within the cell, processes that underlie the onset of tolerance. It has also been shown that after internalization within the cell, the phosphorylation pattern governs the eventual fate of the recptor. This includes pathways that lead to degradation of the receptor, recycling to the cell surface, or interfacing with other messenger pathways. Moreover, there is evidence that bound forms of arrestin may differ depending on its binding partner. The biochemical evidence thus leads to the hypothesis that alternate complexes of the arrestin with the GPCR governs the eventual fate of the receptor. Thus it is our assertion that arrestin is an intriguing drug target and the modulation of its function offers the potential to control the fate of the receptor after it has been activated. This proposal seeks to develop arrestin-2 as a drug target, establish techniques for Nuclear Magnetic Resonance Based Screening of ligands targeted to arrestin-2 and validate these techniques against a library of compounds optimized for chemical diversity. These specific aims will accomplish efficient sampling of ligands across a range of affinities from lower affinity to high affinity (10-3 - 10-8 M). This offers a double benefit in allowing the sampling of a greater range of chemical diversity and allowing a more extensive mapping of potential binding sites that may be exploited to modulate the action of arrestin-2. The notion-of targeting arrestin-2 as a means to modulate biochemical pathways that may affect addiction is a novel hypothesis and the development of ligands that accomplish this would offer an invaluable tool to determine the impact that these biological processes have on addiction. The establishment of such lead compounds would further serve the design and development of therapeutic agents that circumvent tolerance and hence potentially treat the symptoms of drug addiction. PUBLIC HEALTH RELEVANCE: This knowledge will benefit human health by establishing methods for screening of ligands against arrestin-2. The characterization of arrestin2 active sites and tools for the discovery of ligands targeted to these sites will be valuable in evaluating how arrestin contributes to GPCR signaling. The GPRC-arrestin interaction mediates many biological events such as desensitization, internalization and the interaction of GPCR's with multiple messenger systems. These events underly many biological processes including drug tolerance and addiction. This work will lead to the development of tools to better understand these processes and may contribute to the design of therapeutic agents that help to treat addiction and circumvent tolerance to therapeutic drugs.